Systems and methods for the detection and analysis of free thiol

ABSTRACT

Embodiments described herein relate to devices, and methods for quantifying thiol content in a sample containing a mixture of proteins or protein isoforms. The method includes conjugating a portion of the sample with free thiol detection binders, separating the contents in the portion of the sample into separated protein isoforms, detecting fluorescence signals associated with each separated protein isoform, and quantifying, based on the fluorescence signals, a relative amount of free thiol associated with each separated protein isoform. In some instances, the method includes quantifying the amount of each separated protein isoform based on absorbance signals associated with each separated protein isoform. In some instances, the fluorescence and/or absorbance signals associated with protein isoforms conjugated with detection binders can be compared with the corresponding signals associated with unconjugated protein isoforms. In some instances, the method further includes applying a reducing agent and quantifying total-thiol content in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/US2019/047965, filed Aug. 23, 2019, which claims priority to andthe benefit of U.S. Provisional Application No. 62/722,012, filed Aug.23, 2018, entitled “Systems and Methods for the Detection and Analysisof Free Thiol,” the disclosure of each of which is incorporated hereinby reference in its entirety.

FIELD

Some embodiments described herein relate to systems, apparatuses andmethods for the detection and analysis of free thiol in samples ofbiological materials.

BACKGROUND

A thiol is an organosulfur compound that includes a carbon bondedsulfhydryl (R—SH) group, R being an alkyl or aromatic or similarsubstituent. Thiols, also referred to as free-thiols and/or free thiolgroup(s), are the sulfur analogs of alcohols with sulfur taking theplace of the oxygen in the hydroxyl group (—OH) of the alcohol. Thiolscan also be generated by the reduction of compounds including one ormore disulfide bridges or disulfide groups, which become free thiolsafter the reduction and contribute to the total thiol content of thecompound.

Thiols play a significant role in several naturally occurring andsynthetic bio-organic molecules. Free thiols in biological systems haveimportant regulatory roles. For example, oxidatively modified thiolgroups of cysteine residues are known to modulate the activity of agrowing number of proteins. As another example, disulfide bond formationis a key posttranslational modification, with implications forstructure, function and stability of numerous proteins. While disulfidebond formation is a necessary and essential process for many proteins,it is deleterious and disruptive for others. Cells regulatethiol-disulfide bond homeostasis, through Thiol-disulfide exchangereactions, that play critical roles in many aspects of cellularfunction. The detection and measurement of free thiols (e.g., thiolsassociated with free cysteine, glutathione, and cysteine residues onproteins) is an important tool for investigating biological processesand events in many biological systems.

Thiol groups can be present on various proteins, for example as part ofcysteine residues included in the various proteins. Thiol groups canalso be present on various protein isoforms, a set of highly similarproteins that perform the same or similar biological roles. A set ofprotein isoforms may be formed from alternative splicings or otherpost-transcriptional modifications of a single gene. Thiol groups may bepresent on synthetically derived products such as Antibody-drugconjugates (ADCs), bioconjugates, and immunoconjugates and otherbiopharmaceutical drugs designed as a targeted therapy for treatment ofvarious conditions (e.g., treatment of various cancers). ADS, forexample, combine the specificity, favorable pharmacokinetics, andbiodistributions of antibodies with the destructive potential of highlypotent drugs. For example, in some instances, ADCs can be designed tocombine the targeting capabilities of monoclonal antibodies with thecancer-killing ability of cytotoxic drugs designed to discriminatebetween healthy and diseased tissue. Thus, unlike chemotherapy, ADCs cantarget and kill tumor cells while sparing healthy cells. ADCs are formedby linking or conjugating specific antibodies to specific therapeuticdrugs. Thiol groups present on the antibodies can be used as linkagesites to conjugate the drug to form ADCs. In some instances, ADCs caninclude an antibody linked via thiol groups to a biologically activecytotoxic (anticancer) payload or drug.

Thiols are typically detected directly by virtue of their relativelyhigh reactivity compared to most other common species in biologicalsystems. However, significant challenges exist to analyzing thiolcontent in samples such as those mentioned above. For example, currentlyavailable methods of quantification of free thiols lack furtherspecificity such as the specific proteins or protein isoforms that mayinclude free thiol groups (for example included in cysteine residues).In the development of biologic therapeutics knowing, for example, whichisoforms have particular characteristics, such as the presence of freethiol groups, is important to determine the allowable limits for eachisoform.

Increasingly, alternative formats of traditional therapeutic antibodiesare being developed including the use of IgG2 antibodies with additionaldisulfide bonds. For example, such antibodies can be used in thedevelopment of ADC (Antibody Drug Conjugates), bispecific antibodies,multispecific antibodies, and other multivalent and/or conjugateproducts. Furthermore, some products may involve the reduction ofantibody halves before reassembly of the final product. Additionally,the antibody for ADC products typically have engineered cysteines forattachment of the drug conjugate linker. These processes can lead toincreased free thiol heterogeneity. As a result, it can be beneficial toanalyze such isoforms. Known techniques for detecting free thiolcontent, however, are generally inadequate to detect free thiol contentof such antibodies. For example, known techniques for quantifying thiolcontent involve testing portions of the sample with one marker at atime, which involves significant time and expense. As another example,some known methods of measuring thiol content are unable to distinguishbetween free thiols and disulfide bridges.

Methods to objectively identify and quantify free thiol and total thiolcontent in a biological sample, with specificity to individual portionsof the sample such as protein isoforms are currently lacking. A needtherefore exists for methods and apparatus for deconvolvingprotein-containing samples and analyzing the deconvolved or separatedsamples for free thiol and/or total thiol content.

SUMMARY

Some embodiments described herein relate to systems, apparatuses andmethods for differential analysis of a sample containing of biologicalmaterials, such as protein isoforms that include thiol groups and/ordisulfide bridges.

In some embodiments, a method includes generating, from a samplecontaining a plurality of protein isoforms at least some of which haveone or more free thiol groups, a first sample portion and a secondsample portion. The method includes applying, to the first sampleportion, a fluorescent detection binder configured to bind to free thiolgroups to produce conjugated thiols, without reducing the plurality ofprotein isoforms included in the first sample portion. The methodincludes separating the plurality of protein isoforms in the firstsample portion to generate a first set of separated protein isoforms,and separating the plurality of protein isoforms in the second sampleportion to generate a second set of separated protein isoforms. Themethod includes detecting a first fluorescence signal associated withthe conjugated thiols and an absorbance signal associated with the firstset of separated protein isoforms. The method further includesidentifying, based on the absorbance signal, at least one of a quantityor an identity of each separated protein isoform from the first set ofseparated protein isoforms. The method also includes detecting a secondfluorescence signal associated with the second set of separated proteinisoforms, and measuring, based on a difference between the firstfluorescence signal and the second fluorescence signal, a quantity offree thiol groups associated with each separated protein isoform fromthe first set of separated protein isoforms.

In some embodiments, a system includes a memory and a processoroperatively coupled to the memory. The processor is configured toreceive a fluorescence signal associated with a first set of separatedprotein isoforms. The first set of separated protein isoforms can beobtained from a first portion of a sample containing a plurality ofprotein isoforms, at least some protein isoforms from the plurality ofprotein isoforms having one or more free thiol groups. The fluorescencesignal can be associated with a fluorescent detection binder applied tothe first sample portion that is configured to bind to free thiolgroups. The processor can be configured to receive a first absorbancesignal associated with the first set of separated protein isoforms, andreceive a second absorbance signal associated with a second portion ofthe sample. The processor can be further configured to identify, basedon the first absorbance signal and the second absorbance signal, atleast a subset of the protein isoforms from the sample, and calculate,based on the fluorescence signal, a quantity of free thiol groupsassociated with each protein isoform from the subset of the proteinisoforms.

In some embodiments, a method includes applying, to a sample containinga plurality of protein isoforms, a set of fluorescent detection binders.The set of fluorescent detection binders can be configured to bind tofree thiols without reducing protein isoforms. The method includesseparating the sample via isoelectric focusing to generate a set ofseparated protein isoforms. The method further includes obtaining afluorescence signal and an absorbance signal associated with the set ofseparated protein isoforms, identifying, based on the absorbance signal,at least one of a quantity or an identity of each separated proteinisoform from the set of separated protein isoforms; and measuring, basedon the fluorescence signal, a quantity of free thiols associated witheach separated protein isoform from the set of separated proteinisoforms.

In some embodiments, a method includes processing at least a portion ofa sample containing a mixture of proteins and/or protein isoforms. Themethod includes preparing a sample potentially containing thiols anddividing the sample into a first and second portion. The method includesconjugating the first portion of the sample with one or more specificdetection binders targeting free thiol groups to produce conjugatedthiols. The method includes separating the proteins or protein isoformsin the first and second portions of the sample and performingfluorescence emission measurements on the separated contents of thefirst and second portions of the sample. The method further includescomparing the fluorescence measurements of the first portion of thesample, including separated proteins or protein isoforms conjugated withdetection binders, against fluorescence measurements of the secondportion of the sample, including separated proteins or protein isoformsimilar to that of the first portion but unconjugated with detectionbinders. The method further includes quantifying the amount of freethiol content in the separated contents of the sample based on thecomparison of fluorescence measurements obtained from the separatedcontents of the first and second portions of the sample, describedabove.

In some instances, in addition to the fluorescence measurements, themethod optionally includes performing absorbance measurements (e.g.,ultraviolet (UV) absorbance measurements) from the separated contents ofthe first and second portions of the sample, and comparing theabsorbance measurements of the separated contents of the first andsecond portions of the sample. In such instances, based on thecomparison of the absorbance measurements, the method includesquantifying the amount of the separated protein or protein isoform bydeconvolving and contribution of the conjugated detection binder to theabsorbance (e.g., increased UV absorbance from the conjugatedfluorescent dye or antibody acting as the detection binder).

In some instances, the method further includes evaluating whether thetotal thiol content of the sample is in the form of free thiol. Forexample, if the sample may include thiol and disulfide groups, themethod includes determining whether there may be disulfide bridges inthe sample. If the sample is determined to potentially contain disulfidegroups, the method includes converting any potential disulfide groupsinto thiol groups using suitable methods, and conducting free thioldetection following the procedure described above, and in further detailherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a system configured to performdifferential analysis for quantifying free thiol content in a samplecontaining mixtures of proteins and/or protein isoforms, according to anembodiment.

FIG. 2 is a flowchart of a method to perform a differential analysis forquantifying free thiol content in a sample containing a mixture ofproteins and/or protein isoforms, according to an embodiment.

FIG. 3 is a flowchart of a method to perform a differential analysis forquantifying free thiol content in a sample containing a mixture ofproteins and/or protein isoforms, according to an embodiment.

FIG. 4A is a chart showing a plot of an example absorbance signaldetected from a sample containing a plurality of proteins and/or proteinisoforms, plotted as a function of isoelectric point, using a system asdescribed herein, according to an embodiment.

FIG. 4B is a chart showing a plot of an example fluorescence signaldetected from the sample containing a plurality of proteins and/orprotein isoforms shown in FIG. 4A, plotted as a function of isoelectricpoint, using a system as described herein, according to an embodiment.

FIG. 5 is a chart showing an overlay of the absorbance signal of FIG. 4Aand the fluorescence signal shown in FIG. 4B, indicating a relationshipbetween peaks observed in each signal with the peaks observed in theother signal, the peaks being associated with a set of plurality ofproteins and/or protein isoforms of the plurality of proteins and/orprotein isoforms, as analyzed using a system as described herein,according to an embodiment.

DETAILED DESCRIPTION

Systems, methods and apparatuses of the disclosure relate to thequantification and differential analysis of free thiol content in asample containing a mixture of proteins and/or protein isoforms.

FIG. 1 shows a schematic of an example Free Thiol Analysis (FTA) system100. The FTA system (also referred to here as “the analysis system” orsimply “the system”) is operable to perform a differential analysis of asample containing a mixture of proteins and/or protein isoforms toquantify the free thiol content. Performing differential analysis caninclude (i) separation of the contents of the sample, such as proteinsand/or protein isoforms, using any suitable separation method, and (ii)using a combination of one or more types of detection binders thatspecifically bind to free thiol groups present on the proteins orprotein isoforms in the sample to produce conjugated thiols.

The system 100 includes a sample handler 102, a sample prober 104, asignal recorder 106, and a signal analyzer 110 (also referred to as thedifferential analyzer or simply the analyzer). The signal analyzer 110can be any suitable computing entity having a processor 120 and a memory140. For example, the signal analyzer can be a personal computer, alaptop computer, an enterprise system at least partially hosted in anenterprise server, such as, for example a web server, an applicationserver, a proxy server, a telnet server, a file transfer protocol (FTP)server, a mail server, a list server, a collaboration server and/or anyother suitable computing entity.

The various components of the system 100 can be interconnected in anysuitable manner (physically, fluidically, and/or electrically, e.g.,through wired or wireless connection methods). In some embodiments, thesignal analyzer 110 can be physically collocated with the sample handler102, sample prober 104 and/or signal recorder 106 (e.g., disposed in thesame room or within a common housing). Although not shown in FIG. 1, thesystem 100 can be configured to be coupled to one or more other externalcomputing entities (e.g., personal computers, servers, cloud servers,etc., which may include a processor and a memory) in any suitable mannerthough a communication network or a communication channel. For example,the signal analyzer 110 can be partially and/or completely implementedusing an external computing entity.

FIG. 2 is a flow chart of a method 200 of performing differentialanalysis to quantify free thiol and/or total thiol in a samplecontaining a mixture of proteins and/or protein isoforms, according toan embodiment. The method 200 can be carried out using a systemdescribed herein (e.g., system 100 described with reference to FIG. 1).In particular constituent proteins or protein isoforms can be separatedand analyzed individually for their thiol content, allowing simultaneousidentification of protein/protein isoforms and the quantification offree thiol content in a protein specific or isoform specific manner.

At 201, a sample containing a mixture or a plurality of proteins and/orprotein isoforms is prepared. The sample can be prepared from anaturally occurring source such as a bodily fluid like plasma, blood,urine, and so forth and/or a tissue-derived sample. The sample may, insome instances, be prepared from a synthetically generated or engineeredsource such as a synthetic and/or recombinant antibody preparation, anAntibody-Drug-Conjugate (ADC), or the like. Preparing the sample caninclude (ultra)centrifugation of the sample, size exclusionchromatography, (ultra)filtration, or any other suitable means ofobtaining a protein fraction or fraction containing protein isoformsfrom the source. In some instances sample preparation can include stepssuch as cell disruption, extraction, solubilization, removal ofinterfering compounds, changes of the physio/chemical properties andconcentration of the sample, etc. In some instances, a sequence of oneor more steps can be followed to prepare the sample based on propertiesof the sample such as type of fluid, source of fluid, medical history ofthe patient providing the sample, amount or quantity of the sample, etc.The prepared sample is then divided into a first portion and a secondportion.

As described in further detail herein, a differential analysis of asample containing a mixture of proteins and/or protein isoforms forquantification of free thiol content can be performed using detectionbinders configured to bind or form complexes with free thiol groups inthe proteins or protein isoforms to produce conjugated thiols. At 203the contents of the first portion of the sample from 201 can beconjugated with detection binders specific for thiol groups to produceconjugated thiols. A mixture containing detection binders can becombined with the first portion of the sample; the mixture can beformulated such that it results in no or minimal reducing effects on thecontents of the first portion of sample.

Detection binders can be, for example, fluorescent dyes configured toselectively bind to thiol groups. For example, fluorescent modificationof proteins using covalent methods can include derivatization of theproteins or proteomic samples with a fluorescent dye prior to anyseparation of the protein contents of the first portion of the sample,as disclosed below. Alternatively, using noncovalent methods, theproteins or isoforms in the first portion of the sample may first beseparated by a suitable separation method (e.g., by capillaryelectrophoresis, SDS-PAGE, etc.) and then the separated protein orisoform bands may be stained with dyes that bind to SDS-proteincomplexes, e.g., the SYPRO dyes. The dyes can be configured to benon-reducing such that the dyes specifically target free thiol groups inthe sample without altering existing disulfide bonds in the sample.

An example of covalently labelling thiol groups of proteins and proteinisoform mixtures with fluorescent dyes at 203 is to label the cysteine(Cys) residues of the proteins and protein isoforms with thiol-reactivedyes, such as iodoacetamide dyes (e.g., BODIPY TMR cadaverine IA andBODIPY F1 C1-IA, -TMRIA and eosin-5-iodoacetamide), or maleimide dyes(e.g., ThioGlo I and Rhodamine Red C2 maleimide). Dyes may be selectedbased on properties such as desired dye concentration, the amount ofthiol content expected, the concentration of the protein and/or proteinisoform content in the first portion of the sample, the specificity ofbinding to thiol groups under the desired dye concentration, etc.

At 205, the second portion of the sample is retained. The second portionof the sample remains unconjugated to any detection binder and isretained for use as a reference sample to analyze the contents of thefirst portion as discussed in further detail herein.

At 207A, the contents of the first portion of the sample, conjugatedwith detection binders (e.g., fluorescent dyes) at 203, are separatedusing any suitable separation method to generate a set of separatedprotein isoforms. In some embodiments, a sample handler (e.g., samplehandler 102) can be used to separate the first portion of the sample togenerate the separated protein isoforms. For example, proteins withvarying charge, size, mobility, and/or molecular weight may be separatedusing methods like electrophoresis such as imaged capillaryelectrophoresis, SDS-PAGE, or other suitable technique. As anotherexample, proteins and/or protein isoforms (conjugated to detectionbinders) may be separated based on their isoelectric point with methodslike (capillary or gel-based) Isoelectric Focusing (IEF). Afterseparation, the contents of the first portion of the sample may be inthe form of separated bands of proteins and/or protein isoforms,optionally immobilized in a medium such as a gel or capillary. In someinstances, as disclosed above, the proteins and/or protein isoforms canfirst be separated (e.g., as described at 207A of flowchart 200) and theseparated bands of proteins and/or protein isoforms can be conjugatedwith non-covalent dyes (e.g., as described at 203 of flowchart 200).

At 207B, the contents of the second portion of the sample (the portionthat is unconjugated with detection binders) is made to undergo asimilar separation procedure as the first portion of the sample at 207AAfter separation, the contents of the second portion of the sample maybe in the form of separated bands of proteins and/or protein isoforms,optionally immobilized in a medium such as a gel or capillary. Forexample, a sample handler (e.g., sample handler 102) can separate thesecond portion of the sample into separated protein isoforms. In someimplementations, sample handling and/or separation can be done in aplate format. In some embodiments, the separation of the contents of thefirst portion of the sample and the contents of the second portion ofthe sample can be performed simultaneously and/or in parallel. Forexample, the first portion of the sample and the second portion of thesample can be separated in parallel capillaries or gel-lanes defined ina sample handler (e.g., sample handler 102 described herein) during asingle separation run. In other embodiments, the contents of the firstportion of the sample and the second portion of the sample can beseparated in different/sequential separation runs.

At 209A, the bands of separated proteins and/or protein isoforms of thefirst portion of the sample, conjugated to detection binders, aredetected via a fluorescence emission measurement. In some embodiments, asample prober (e.g., sample prober 104) can be used to induce and/ormeasure fluorescence emission from the conjugated detection binders. Forexample, light of a suitable wavelength (e.g., selected to match theexcitation spectrum of the detection binders used) and suitableintensity may be used to probe the medium containing the separated bandsof conjugated proteins or protein isoforms, for a predetermined periodof time (also referred to herein as “exposure”) and excite theconjugated fluorescent dyes, using suitable fluorescent imagingtechniques. Fluorescence signals may be emitted in response to theexcitation light probe. Because the detection binders selectively bindto thiol groups, the fluorescence signals can be associated with theconjugated thiols. The emitted fluorescence signals, which can becorrelated to thiol-groups in the separated bands of conjugated proteinsand isoforms, are detected, recorded and/or stored for further analysis.In some embodiments, the emitted fluorescence signals can be detectedand/or quantified using a signal recorder (e.g., signal recorder 106) toobtain fluorescence measurements. In some instances, the detectedfluorescence signals can be analyzed and transformed into a suitablerepresentation, for example representations in the form of graphs ofsignal intensity as a function of time of recording or as graphs ofsignal intensity as a function of position of scanning. In someinstances, the recorded fluorescence signals may be transformed into aform of a spatial map of the medium in two or three spatial dimensions,with or without a temporal dimension corresponding to time of recordingof the signals. In other words, the representation of the medium can insome instances be an image or a three-dimensional stack of images, eachimage corresponding to a single instance capture of a two- orthree-dimensional view of the medium with pixels corresponding topositions along predetermined axes on the medium. The representation ofthe medium can in other instances be a video of a two- orthree-dimensional spatial region captured over a time period. Therepresentation of the medium can be include the separated bands ofconjugated proteins and/or protein isoforms, with fluorescence intensityvarying as a function of thiol content.

At 209B, the bands of separated proteins or protein isoforms of thesecond portion of the sample, unconjugated to detection binders, areprobed using similar techniques as those discussed above with referenceperforming fluorescence emission measurements at 209A. In someinstances, a native fluorescence (or auto-fluorescence) signal can becaptured from the separated proteins or protein isoforms of the secondportion of the sample, unconjugated to detection binders. In someembodiments, a sample prober (e.g., sample prober 104) can be used toinduce and/or measure native fluorescence emission from the conjugateddetection binders. For example, light of the same wavelength orfrequency and intensity used to induce fluorescence emission may be usedto probe the medium containing the separated bands of unconjugatedproteins or protein isoforms, using the same or similar fluorescentimaging techniques as in 209A. The signals in response to the lightprobe can be recorded and stored in a manner similar to that performedat 209A. In some embodiments, the emitted native fluorescence signalscan be detected and/or quantified using a signal recorder (e.g., signalrecorder 106) to obtain native fluorescence measurements. For example,the signals in response to the excitation light probe may be an imagecontaining a spatial map of the medium containing the separated bands ofconjugated proteins and/or protein isoforms. However, unlike the resultsat 209A, the map obtained at 209B may lack any significant fluorescenceintensity that corresponds to an indication of thiol content. Themeasurement of fluorescence emission at 209A and 209B may be conductedsequentially or in a substantially parallel manner.

At 213, the fluorescence measurements recorded from the separatedcontents of the first portion (conjugated with fluorescent dyes) at209A, and the fluorescence measurements obtained from the separatedcontents of the second portion (unconjugated) of the sample (alsoreferred to as native fluorescence) are compared. The relative amount offree thiol content in the sample is determined at 217 based on thecomparison performed at 213. For example, in some instances the relativeamount of free thiol content in the sample can be determined based on adifference between the fluorescence measurements recorded from theseparated contents of the first portion conjugated with florescentdetection binders and the native fluorescence measured from theseparated contents of the second portion unconjugated with detectionbinders. In some instances, as described above, the results from 209Aand 209B may be in the form of images containing spatial maps of theseparated proteins and/or protein isoforms, with corresponding locationsof the medium at 209A and 209B (e.g., locations in a capillary, on aSDS-PAGE gel, or a IEF medium) containing the same protein or isoform inthe conjugated form (209A) and in the unconjugated form (209B),respectively. Thus, the resulting fluorescence image containing spatialmaps of the medium from 209A and 209B may contain fluorescencesignatures indicating the presence and absence of fluorescent dyes onthe same proteins or protein isoforms, respectively. In some instances,depending on the detection binders used to bind free thiol in the firstportion of the sample, there may be shifts or differences in therelative locations of the separated bands of conjugated proteins orisoforms with reference to the locations of the unconjugated proteins orisoforms of the same identity. For example, the detection binders mayalter the mobility, isoelectric point, or other characteristic of theproteins or isoforms. Such shifts may be taken into consideration duringany analysis of results based on the separated bands of proteins orisoforms (e.g., during the analysis of fluorescence emission or UVabsorbance as described below).

As described above, the image from 209A shows fluorescence signalsindicating the locations where specific proteins or protein isoforms arelocated, with the fluorescence intensity corresponding with the amountof thiol content. Whereas, the image from 209B, taken of the secondportion of the sample, which lacks thiol-specific fluorescent dyes, isexpected to show reduced or minimal fluorescence signals at thelocations where specific proteins or protein isoforms are expected to beimmobilized (with fluorescence detected being caused byauto-fluorescence or background materials in the sample or medium thatmay cause some non-zero amount of fluorescence emission). Thus,fluorescence measured in the background condition from 209B can be usedas a reference to quantify the amount of thiol content. The fluorescencemeasured in the background condition from 209B can be subtracted from(or otherwise used to correct) the intensity of fluorescence accountedfor by the presence of fluorescence dyes in the results from 209A. Inother embodiments, the relative amount of free thiol content in thesample can be determined based solely on the fluorescence measured at209A.

As indicated in the flowchart 200, in some instances the method includesmeasurement of an absorbance signal (e.g., signal associated with UVabsorbance). At 211A, the separated contents of the first portion of thesample, conjugated with fluorescent dyes, is probed with suitablestimulus such as UV light, and the resulting absorbance signal (alsoreferred to herein as “absorbance”) is measured. In some embodiments, asample prober (e.g., sample prober 104) can be used to probe theseparated protein isoforms of the first portion of the sample with UVlight and a signal recorder (e.g., signal recorded 106) can be used tomeasure relative absorption of UV light associated with each separatedprotein isoform of the first portion of the sample. Similarly, at 211B,the same light or a similar stimulus at a similar intensity is used andthe amount of absorbance by the separated contents of the second portionof the sample can be recorded. In some embodiments, a sample prober(e.g., sample prober 104) can be used to probe the separated proteinisoforms of the second portion of the sample with UV light and a signalrecorder (e.g., signal recorded 106) can be used to measure relativeabsorption of UV light associated with each separated protein isoform ofthe second portion of the sample. In some implementations, the separatedprotein isoforms from the first portion and the second portion can beprobed simultaneously and the relative UV absorbance associated witheach separated protein isoform of both portions can be recordedsimultaneously.

As described with reference to results from 209A and 209B, the resultsfrom 211A and 211B can be in the form of images containing spatial mapsof UV absorbance corresponding to different separated proteins and/orprotein isoforms. The measurement of absorbance at 211A and 211B may beconducted sequentially or in a substantially parallel manner.

In some implementations, the fluorescence measurements described at 209Aand 209B, and the UB absorbance measurements described at 211A and 211Bcan be performed simultaneously using one or more sample probers (e.g.,sample prober 104) and one or more signal recorders (e.g., signalrecorder 106). For example, a system may be configured such that asingle sample prober (e.g., UV light source) can induce bothfluorescence emission as well as UB absorbance. As another example, asignal recorder can include one or more detection arms configured todetect and/or record fluorescence signals and UV absorbance signals fromthe separated protein isoforms from the first and/or second portions ofthe sample, simultaneously.

The absorbance signal can be used to identify a quantity and/or anidentity of each separated protein and/or protein isoform from the setof separated proteins/protein isoforms. At 215, the absorbancemeasurements of the separated contents of the first portion is comparedagainst the absorbance of the separated contents of the second portion.At 219, based on the comparison at 215, the amount and/or identity of aspecific protein and/or protein isoform. For example, an increased UVabsorbance may be observed at 211A, and the increased absorbance maycorrespond to the conjugated dye. Thus, the increased UV absorbanceassociated with the conjugated fluorescent dye can be accounted for, andthe amount of the particular isoform can be determined. For example, UVabsorbance can be used to determine relative and/or absolute quantitiesand/or concentrations of protein isoforms. The position of peaksassociated with absorbance signals of proteins and/or protein isoformsobserved at 211A and/or 211B can be used to identify the proteins and/orprotein isoforms, for example, by isoelectric point, molecular weight,etc.

In some instances, the measurements of fluorescence emission (at 209A,209B) and the measurements of UV absorbance (211A, 211B) may beconducted in a sequential manner with the former following the later orvice versa. In some other instances, the measurements of fluorescenceemission (at 209A, 209B) and the measurements of UV absorbance (211A,211B) may be conducted in a substantially parallel manner. In someinstances, the measurement of fluorescence emission (at 209A, 209B) andthe measurement of UV absorbance (211A, 211B) can be conductedsubstantially simultaneously. For example, an absorbance light sourceand a fluorescence excitation light source can illuminate the samplesimultaneously and/or in sequence, in real time—while the proteinsand/or protein isoforms are being separated. In some implementations,the absorbance light source and the fluorescence excitation light can bea single light source emitting light in the UV range (e.g., 280 nm). Inother implementations, the absorbance light source and the fluorescenceexcitation light can be different light sources and/or emit light atdifferent wavelengths. Similarly, the comparison (at 213) andquantification of free thiol content (at 217) and the comparison (215)and quantification/identification of proteins and/or protein isoforms(at 219), can be conducted in a sequential manner (with the formerfollowing the latter or vice versa) or substantially parallel manner.Thus, in some embodiments, a single run containing the first portion ofthe sample and the second portion of the sample (e.g., in differentlanes or capillaries) can be analyzed for fluorescence and absorbance.

At 221, the method includes querying if a test for the total thiolcontent of the sample (and/or the total thiol content of one or moreseparated bands) is indicated. For example, in some instances, a usermay indicate a test for total thiol content of the sample to beperformed. In such instances, the method 200 can be used to test and/orquantify, in addition to the thiol groups in the form of free thiol, ifthere remain functional groups that may be converted to thiols. Forexample, the method at 221 may determine that the total thiol content isof the sample is to be quantified, as the sample may include disulfidegroups that may be converted to thiols. In some instances, such adetermination may be made following 201 to 219 having been carried outwith a given sample without reducing the sample. In some instances, thetest for total thiol content may be indicated and performed withoutperforming the steps 201 to 219, i.e., without quantifying relative freethiol content.

If a test and/or quantification of total thiol content is positivelyindicated at 221, at 223 disulfide groups can be converted to free thiolgroups using suitable procedures. For example, treating the first andsecond portions of the sample with suitable reductants of suitableconcentration may convert the disulfide bonds to free thiol groups.Following which the process outlined in the flowchart 200 from 203 and205 (for each of the first and second portions of the sample) to 217 and219 can repeated to determine a total thiol content (e.g., on aper-band/per-isoform basis). A difference in detected thiol contentduring a second examination of the first and second portions of thesample after reducing sample can be used to determine the absoluteand/or relative quantities and/or concentration of converted thiolcontent (e.g., quantifying disulfide groups).

In some instances, the total thiol in a sample containing free thiol anddisulfide groups that can be converted to thiol groups can be quantifiedas described above, by following the procedure outlined in the flowchart200 in two runs, a first run configured to determine the free thiolcontent in the sample and the second run configured to determine thetotal thiol content, including the converted thiol groups (e.g., thiolgroups formed by reducing disulfide groups). In some instances, thequantification of free thiol and total thiol content can be accomplishedin a single run by having four portions of the sample examinedconcurrently. For example, portions A and B can be obtained from asource of proteins and/or protein isoforms without reduction ofpotential disulfide bonds, and portions C and D can be obtained from thesame source of proteins and/or protein isoforms and subjected to asuitable reducing agent to convert disulfide groups into free thiols.Thiols in the four portions A, B, C, and D (on a per-band and/orper-isoform basis) can be quantified using the procedure outlined in theflowchart 200 in FIG. 2, with portions A and B forming a first set offirst and second portions, respectively, and portions C and D forming asecond set of first and second portions, respectively. In other words,the portions A and C can be treated as the first portion in flowchart200, and the portions B and D can be treated as the second portion inflowchart 200, as described at steps 201-211. Thus, fluorescenceemission measurements and/or absorbance measurements can be obtained forup to all four portions of the sample, A, B, C, and D. At 213, thefluorescence emission measurements of portion A is compared to that ofB, and the fluorescence emission measurements of portion C is comparedto that of D. At 217, based on the fluorescence emission measurementcomparison between portions A and B, the relative amount of free thiolin the unreduced portions, A and B, is quantified. Additionally, at 217,based on the fluorescence emission measurement comparison betweenportions C and D, the total amount of thiol in the reduced portions, Cand D, is quantified. Then, at 225, the quantified amount of free thiolfrom the portions A and B is compared with the quantified amount oftotal thiol from portions C and D and the amount of converted thiol canbe determined. For example, the difference between the total thiolquantification and the free thiol quantification can be used to deducethe amount of converted thiols present in the reduced portions C and D,which may have resulted from reduction of disulfide bonds in theoriginal source from which comparable portions A, B, C, and D wereobtained. Furthermore, at 215, the absorbance measurements from theportion A can be compared to that from portion B, and the absorbancemeasurements from portion C can be compared to that from portion D. At219, based on the absorbance measurement comparison between portions Aand B, the relative amount of each separated protein and/or proteinisoform in the unreduced portions, A and B, is identified and/orquantified. Additionally, at 219, based on the absorbance measurementcomparison between portions C and D, the relative amount of eachseparated protein and/or protein isoform in the reduced portions, C andD, is identified and/or quantified. The identified and/or quantifiedproteins and/or protein isoforms from the various portions can becompared for confirmation and/or control that the reduced portions (Aand B) are substantially similar to the reduced portions (C and D),accounting for effects of reduction and/or conjugation with binders. Insome instances the identified and/or quantified proteins and/or proteinisoforms from the various portions can be compared for quantification ofthe effects of reduction (e.g., reduction of disulfide bonds) or theeffects of conjugation with suitable binders (e.g., antibodies).

The processes described above can also be used, in some instances, toquantify total thiol in a sample without the quantification of freethiol, by using already reduced samples as the first and second portionsdescribed in flowchart 200. Furthermore, in some instances, thefluorescence measurements can be carried out without absorbancemeasurements. Similarly, in some instances, absorbance measurements ofthe first and second portions can be carried out without thefluorescence measurements.

As described previously, the processes described above can be conductedin a system described herein such as the system 100 described above. Insome instances, one or more of the steps in method 200 can be conductedfor several samples in a parallel manner, for example using a multi-wellplate to hold sample portions, etc. In some instances, one or more stepsof the method 200 can be omitted. In some instances, the steps of themethod 200 can be performed in any suitable order. In some instances,the method 200 can include other steps not shown in FIG. 2 to performthe quantification of free thiol and/or total thiol in a samplecontaining a mixture of proteins and/or protein isoforms, as describedin the method 200.

FIG. 3 is a flow chart of an example method 300 of performingdifferential analysis to quantify free thiol in a sample containing aplurality of proteins and/or protein isoforms, according to anembodiment. In some instances, portions of the method 300 can besubstantially similar to the method 200 described above and can carriedout using a system described herein (e.g., system 100 described withreference to FIG. 1). In particular, proteins or protein isoforms can beseparated and analyzed for their thiol content, allowing simultaneousidentification of protein/protein isoforms and the quantification offree thiol content in a protein specific or isoform specific manner.

At 301, one or more fluorescent detection binder can be applied to asample containing a proteins and/or protein isoforms. Fluorescentdetection binder(s) can be configured to bind to free thiols withoutreducing proteins and/or protein isoforms. As described previously withreference to the method 200, the sample can be prepared from a naturallyoccurring source such as a bodily fluid like plasma, blood, urine, andso forth and/or a tissue-derived sample. The sample can be prepared froma synthetically generated or engineered source such as a syntheticand/or recombinant antibody preparation, an Antibody-Drug-Conjugate(ADC), or the like, via any suitable process including cell disruption,extraction, solubilization, removal of interfering compounds, changes ofthe physio/chemical properties and concentration of the sample,(ultra)centrifugation, size exclusion chromatography, (ultra)filtration,or any other suitable process of obtaining a protein fraction orfraction containing protein isoforms from the source. In some instances,as described with reference to method 200, the sample can be dividedinto two or more portions and treated separately. The fluorescentdetection binders can be substantially similar to the fluorescentdetection binders described with reference to the method 200 (e.g.,fluorescent detection binders described as used at 203 of method 200).For example, the fluorescent detection binders can be, fluorescent dyesconfigured to selectively bind, via covalent or non-covalent bonds, tofree thiol (—SH) groups, be excited by light of a particularpredetermined first wavelength or first band of wavelengths, and emitlight at a predetermined second wavelength or second band orwavelengths. As described previously, the fluorescent detection binderscan be configured to be non-reducing such that the dyes specificallytarget free thiol groups in the sample without altering existingdisulfide bonds in the sample. In some implementations, the fluorescentdetection binders can be configured to not altering a chargeheterogeneity associated with a plurality of protein isoforms such thatthe plurality of protein isoforms can be separated using methods thattake advantage of the charge heterogeneity. For example, the fluorescentdetection binders can be configured to not shift the isoelectric pointsof a plurality of protein isoforms such that a separated protein isoformcan be identified based on its isoelectric point pI. In someimplementations, the fluorescent detection binders can be configured tobe UV excitable such that exposure to light of a single wavelength inthe UV range (e.g., 280 nm) can be used to obtain the fluorescencesignal as well as the absorbance signal described below.

At 303, the method 300 includes separating the sample via isoelectricfocusing (or any other suitable technique) to generate a set ofseparated protein isoforms. In some instances, the sample can beseparated into a set of separated protein isoforms via imaged capillaryisoelectric focusing (icIEF), each separated protein isoform includingone or more free thiols. In some instances, the fluorescent detectionbinders can be configured to not modify specific properties of theplurality of protein isoforms such that each protein isoform of theplurality of protein isoforms can be identified even when conjugatedwith the fluorescent binder. For example, in some implementations, thefluorescent binders can be configured to bind to free thiols withoutaltering a charge distribution associated with a plurality of proteinisoforms such that the plurality of protein isoforms bound by thefluorescent detection binders can be separated and/or identified using asuitable method.

As an example, the protein isoforms can be separated to generate a setof separated protein isoforms of the plurality of protein isoforms usingimaged capillary isoelectric focusing (IEF). Briefly, in someimplementations, IEF can involve introducing an ampholyte solutionincluding the sample containing a plurality of protein isoforms, boundto fluorescent detection binders, into one or more immobilized pHgradient (IPG) gels. IPGs can be acrylamide gel matrix co-polymerizedwith a pH gradient, which can result in stable gradient across a rangeof pH values. The IPG can be subjected to an applied electric field byapplying a voltage (e.g., a DC voltage by applying a predeterminedcurrent) via a set of electrodes (e.g., an anode and a cathode). Theimmobilized pH gradient can be obtained by the continuous change in theratio of immobilines (weak acids or bases defined by their pK value). Aprotein isoform, introduced in the pH gradient, that is in a pH regionbelow its isoelectric point (pI) can be positively charged and beinduced to migrate toward the cathode (negatively charged electrode). Asit migrates through a gradient of increasing pH, however, the proteinisoform's overall charge can decrease until the protein isoform reachesa pH region that corresponds to its pI. At this point, the proteinisoform can have no net charge and so migration ceases (as there is noelectrical attraction toward either the anode or the cathode). As aresult, the protein isoforms can become separated protein isoformsfocused into sharp stationary bands with each protein isoform positionedat a point or location in the pH gradient corresponding to its pI. Eachseparated protein isoform can be identified based on the isoelectricpoint (pI) of that protein isoform, the isoelectric point being based onthe charge heterogeneity or charge distribution associated with thatseparated protein isoform.

At 305 the method 300 includes obtaining a fluorescence signal and anabsorbance signal associated with the set of separated protein isoforms.The fluorescence signal can be associated with the fluorescent detectionbinders configured to bind to free thiols. Said in another way, thefluorescence signal can be associated with the conjugated thiolsproduced by a fluorescent detection binder binding to free thiol groupson the separated protein isoforms. As described previously, thefluorescent signal can be obtained by exciting the fluorescent detectionbinders bound to the separated protein isoforms with a light of suitablewavelength to induce fluorescent radiation emission of a particular bandof wavelengths (e.g., 458 nm). In some implementations, the system caninclude one or more optical filters (e.g., a 458 nm long-pass opticalfilter configured to allow passage of light of wavelength greater than458 nm while blocking wavelengths less than 458 nm or any other suitablepredetermined band of wavelengths) to isolate and obtain a fluorescencesignal of the band of wavelengths of interest. In some instances, themethod 300 can include one or more steps (not shown in FIG. 3) ofobtaining a native fluorescence signal from a portion of the sample(e.g., a portion of the sample not conjugated with any fluorescentdetection binders). In some instances, the fluorescence signalassociated with the fluorescent detection binders configured to bind tofree thiols can be compared with the native fluorescence to determinethe quantity of free thiols as described below.

The absorbance signal can be obtained by illuminating the separatedprotein isoforms bound by light of a suitable wavelength (e.g., UV lightat 280 nm) that is known to be differentially absorbed by the separatedproteins isoforms. In some implementations, the system can include oneor more optical filters (e.g., a short-pass optical filter configured toallow passage of light of wavelength less than or equal to 280 nm whileblocking wavelengths greater than 280 nm or any other suitablepredetermined band of wavelengths) to isolate and obtain an absorbancesignal of the band of wavelengths of interest (e.g. 280 nm). In someimplementations, light of a single wavelength (e.g., UV light of 280 nmwavelength) can be used to obtain the UV absorbance signal as well asexcite obtain the fluorescence signal

At 307, the method 300 includes identifying, based on the absorbancesignal, at least one of a quantity or an identity of each separatedprotein isoform from the set of separated protein isoforms. Theabsorbance signal can include a differential UV absorbance associatedwith each separated protein isoform from the sample, which can be usedto identify each separated protein isoform. For example, a differentialUV absorbance associated with a set of separated protein isoforms can bebased on the amino acid composition of each separated protein isoformwith the one or more amino acids included in each separated proteinisoform contributing differentially to the net UV absorbance of thatseparated protein isoform. In some implementations, each separatedprotein isoform can be identified based on the differential UVabsorbance associated with that separated protein isoform. In someimplementations, the intensity of absorbance signal can be proportionalto the amino acid composition of the separated protein isoform. In someimplementations, the intensity of absorbance signal can be associatedwith one or more peaks each peak corresponding to a separated proteinisoform. The intensity of each peak can be associated with the identityof the separated protein isoform that the peak corresponds to.

In some implementations, the fluorescent detection binders conjugatedwith each separated protein isoform can contribute to an absorbancesignal, which can result in an increased absorbance associated with thecombination of the separated protein isoform and the conjugatedfluorescent detection binder. In some such implementations, a secondabsorbance signal can be obtained (not shown in FIG. 3) from a secondset of separated protein isoforms from a second portion of the sample(e.g., a portion of the sample not conjugated with any fluorescentdetection binders). The first absorbance signal from separated proteinisoforms conjugated with fluorescent detection binders can be comparedwith the second absorbance signal without the contribution of thedetection binders to determine the quantity or the identity of eachseparated protein isoform.

At 309, the method 300 includes measuring, based on the fluorescencesignal, a quantity of free thiols associated with each separated proteinisoform from the set of separated protein isoforms. In someimplementations, the intensity of fluorescent radiation emitted can beproportional to the number of fluorescent binders, which can be based onthe number of free thiols associated with each separated proteinisoform. In some instances, a difference between the fluorescence signalassociated with the fluorescent detection binders bound to free thiolsand a native fluorescence obtained from a portion of the sample notconjugated with any fluorescent detection binders can be used todetermine the quantity of free thiols. In some implementations, theintensity of fluorescence signal can be associated with one or morepeaks each peak corresponding to a separated protein isoform. Theintensity of each peak can be associated with the relative free thiolcontent of the separated protein isoform that the peak corresponds to.

In some implementations, the system can correlate between the measuredquantity of free thiols and the resolved identity of each separatedprotein isoform to return a measure of free thiol associated with eachidentified protein isoform included in the plurality of protein isoformscontained in the sample. In some implementations, the absorbance signaland the fluorescence signal can be obtained-simultaneously orsubstantially simultaneously (e.g., within 10 seconds of each other).For example, the system can include an excitation sources (e.g., lightsource) and an absorbance source (e.g., UV light) that can be operatedsimultaneously to induce fluorescence radiation emission and absorption(e.g. UV absorption) such that the emitted fluorescence signal and theabsorbance signal can be obtained simultaneously and/or substantiallysimultaneously (e.g., by the same or independent optical sensors,optionally equipped with suitable optical filter combinations).

In some implementations, the system described herein can measure thequantity of each separated protein isoform from the set of separatedprotein isoforms. Based on the quantity of each protein isoforms and thecorresponding quantity of free thiol groups associated with that proteinisoform the system can determine, for each separated protein isoform, anumber of free thiol groups associated with that protein isoform.

As described previously, in some implementations, the systems andmethods described herein can be used to determine a total thiol contentof a set of protein isoforms. The proteins and/or protein isoforms of asample can be reduced to generate free thiol groups from disulfidegroups and the determination of a quantity of free thiol groups can becarried out as described above. For example, the quantity of a first setof free thiol groups can be determined without reducing the proteinisoforms in the sample by using a first portion of the sample, asdescribed above. Following which, another second portion of the samplecan be used to determine the total thiol content. The total thiolcontent can be determined by applying, to the second portion of thesample, a reducing agent configured to reduce disulfide groups togenerate a second set of free thiol groups additional to the first setof free thiol groups.

The quantity of total thiol content can be determined by following theprocedure described above with reference to determining the quantity offree thiol groups. For example, the quantity of total thiol can bedetermined by applying fluorescent detection binders to the reducedsecond portion of the sample, separating the plurality of proteinisoforms in the second sample portion to generate a second set ofseparated protein isoforms, detecting a fluorescence signal associatedwith the second set of conjugated thiols and a second absorbance signalassociated with the second set of separated protein isoforms. Thequantity and or identity of each separated protein isoform from thesecond set of separated protein isoforms and the associated total thiolcontent can be determined based on the second absorbance and secondfluorescence signals. As described previously, in some implementations,an unconjugated portion of the reduced portion of the sample can be usedto obtain a native fluorescence signal and/or an absorbance signaldevoid of contribution from the fluorescent binders. A differencebetween a total fluorescence signal, obtained from each separatedprotein isoform from the second sample portion, and the nativefluorescence signal, from each separated protein isoform from the secondsample portion, can be used to calculate an intensity of fluorescencesignal associated with conjugated free thiols produced from bindingfluorescent detection binders to the total thiol content. The absorbancesignal devoid of contribution from the fluorescent binders can be usedto identify the separated protein isoforms in the second portion of thesample.

In some implementations, as described in further detail below (e.g.,with reference to FIG. 5) the examination of a sample containing proteinisoforms with free thiol groups, using systems and methods describedherein, can include an examination of the detection binders (e.g.,fluorescent dyes) in a state that is unbound or unconjugated with anyprotein isoforms, also referred to as “dye blank”. In addition todetection and measurement of signals (e.g., fluorescence and absorbancesignals) associated with detection binders conjugated with proteinisoforms, a system can acquire signals (e.g., fluorescence andabsorbance signals) from the dye blank or the detection binders (e.g.,fluorescent dyes) that are unbound or unconjugated with any proteinisoforms to be used as reference signals. (e.g., reference signals 471and 481 in FIG. 5). The reference signals can be used to ascertain thatno spurious peaks due to artifacts (e.g., not associated with anyprotein isoforms) are detected and/or misidentified. In some instances,the reference signals can also be used to quantify accurate fluorescenceand/or absorbance signals using accurate baseline values. For example, adifference between the fluorescence signal acquired from detectionbinders conjugated to free thiols on protein isoforms and thefluorescence signal acquired from detection binders unbound to anyprotein isoforms can be used as an accurate florescence signal. Adifference between the absorbance signal acquired from detection bindersconjugated to free thiols on protein isoforms and the absorbance signalacquired from detection binders unbound to any protein isoforms can beused as an accurate absorbance signal.

In some instances, the systems and methods described herein can be usedto compute a difference between the quantity of total thiol and thequantity of the first set of free thiol groups, to determine thequantity of second set of thiol groups generated from reducing disulfidegroups in the second portion of the sample Based on the quantity ofsecond set of thiol groups and a quantity of each protein isoform thesystem can calculate a quantity of disulfide groups included in eachprotein isoform of the plurality of protein isoforms included in thesample.

The systems and methods described herein can be used to determine anabsolute quantity of free thiol in a protein isoform. For example, insome instances, the systems and methods described herein can includeapplying the fluorescent detection binder to a standard sample thatincludes a known quantity of free thiol groups to obtain a standardizedfluorescent signal. The fluorescent signal obtained from a separatedprotein isoform including thiol groups (e.g., free thiol groups from anon-reduced sample or a reduced sample) conjugated with the fluorescentdetection binders can be compared to the standardized fluorescent signaland, based on the comparison, an absolute quantity of thiol groups inthe separated protein isoform can be computed. For example, the absolutequantity of thiol groups (free thiol groups and/or total thiol groups)in the separated protein isoform can be computed from a ratio of thefluorescent signal obtained from a separated protein isoform (from anon-reduced or a reduced sample portion) and the standardizedfluorescent signal associated with the known quantity of free thiolgroups. In some instances, a quantity of disulfide groups in the samplecan be measured based on a difference between the absolute quantity oftotal thiol groups and the absolute quantity of free thiol groupsmeasured in a sample. For example, the additional number of thiol groupsin the total thiol groups above the free thiol groups can be attributedto the disulfide groups that were reduced by the application of areducing agent.

As described herein, the systems and methods disclosed can be used todetermine, in a protein isoform specific manner, a quantity of freethiol groups in a sample containing protein isoforms. Further, by usinga reducing agent, the systems and methods disclosed can be used todetermine, also in a protein isoform specific manner, a quantity oftotal thiol and/or a quantity of disulfide groups included in theprotein isoforms contained in the sample. In some instances, thequantified thiol groups and/or disulfide groups can be surface thiols orsurface disulfides. In some implementations, the systems and methodsdescribed herein can be used to determine, also in a protein isoformspecific manner, a quantity of internal thiol groups and/or internaldisulfide groups in a protein isoform.

In some instances, a protein isoform can assume a three dimensionalstructure, for example, according to a conformational state of theprotein isoform. Based on the three dimensional structure, some of thethiol groups and/or disulfide groups included in the protein isoform canbe accessible surface thiol groups and/or surface disulfide groups. Someother thiol groups and/or disulfide groups may be internal thiol groupsand/or disulfide groups, hidden, unexposed and/or inaccessible bydetection binders due to the three dimensional structure or conformationof the protein isoform. In some implementations, the systems and methodsdescribed herein can be used to detect and/or quantify these internal orhidden thiols groups and/or disulfide groups by denaturing the proteinisoform and converting the hidden internal thiol groups into free thiolgroups accessible to detection binders. Following the denaturing, forexample by applying urea or any other suitable denaturing agent, thesystems and methods described herein can be used to determine a quantityof free thiol groups as described previously (e.g., with reference tomethod 200 and/or method 300 described above).

In some implementations, determination of a quantity of free thiolgroups before and after denaturing can be used to calculate a quantityof internal thiol groups. For example, a difference between the quantityof free thiol after denaturing and the quantity of free thiol groupsbefore denaturing can be used to obtain a quantity of internal thiolgroups). In some implementations, a number of free thiols groups and anumber of internal thiol groups can be quantified and based on adetermination of a quantity of each protein isoform, a quantity of freethiols groups and a quantity of hidden thiol groups can be determined.In some implementations, a combination of applying a reducing agent anda denaturing agent can be used to quantify a quantity of free surfacethiol groups, surface disulfide groups, internal thiol groups, and/orinternal disulfide groups. For example, a first reducing agent and adenaturing agent and a second reducing agent can be sequentially appliedto a sample containing protein isoforms, such that the number of freethiol groups is incrementally and/or cumulatively increased by thereducing and the denaturing steps. In some implementations, the firstreducing agent can be configured to convert surface disulfide groupsinto free thiol groups. The denaturing agent can be configured to makeinternal free thiol groups accessible to be conjugated with detectionbinders and/or make internal disulfide groups accessible to a reducingagent. The second reducing agent (which in some implementations can be areapplication of the same reducing agent as the first reducing agent)can be configured to convert the now accessible disulfide groups, whichwere inaccessible before the denaturing, into free thiol groups. Fromdetermining a quantity of free thiol groups before and after applyingthe reducing agent, determining a quantity of free thiol groups beforeand after applying the denaturing agent, determining a quantity of freethiol groups before and after applying the second reducing agent, andcalculating differences between each determined quantity of free thiolgroups and the quantity of free thiol prior to each manipulation, onecan calculate a quantity of surface free thiol groups, surface disulfidegroups, internal thiol groups and/or internal disulfide groups. As anexample, a difference between the quantity of free thiol afterdenaturing and the quantity of free thiol groups before denaturing canbe used to obtain a quantity of internal thiol groups. In someimplementations, the denaturing can be carried in out in one or moresteps using any suitable method (e.g., chemical denaturing agent, heatbased denaturing, pH based denaturing, etc).

FIGS. 4A and 4B illustrate a charts 450 and 460 showing plots of anexample absorbance signal 451 and an example fluorescence signal 461obtained from a Herceptin® Biosimilar sample, which contains a pluralityof proteins and/or protein isoforms including therapeutic proteins,using a system as described herein, according to an embodiment. Theplurality of proteins and/or protein isoforms in the sample associatedwith the absorbance signal 451 and the fluorescence signal 461 can beseparated using imaged capillary isoelectric focusing over a pHgradient. The absorbance signal 451 of FIG. 4A is shown to be plotted asa measure of intensity of absorbance (measured in mAU—milli absorbanceunits) as a function of isoelectric poing (pI). The fluorescence signal461 of FIG. 4B is shown to be plotted as a measure of intensity offluorescence emission (measured in arbitrary counts) as a function ofisoelectirc point (pI).

As shown in FIG. 4A, the absorbance signal 451 includes peaks 452 witheach peak corresponding to a protein isoform (or pI marker, at pI 7.2)having the corresponding pI value. As an example, a relatively largerpeak 452A of absorbance is associated with a protein isoform A with a pIof approximately 8.65 at 453A compared to a relatively smaller peak 452Dof absorbance that is associated with a protein isoform D with a pI ofapproximately 8.9 at 453D. The measurement of the absorbance signal 451at a given pI value corresponding to a specific protein isoform can beused to determine a relative quantity of that specific protein isoform.The differential absorbance between the peaks 452A and 452D can indicatea difference in the constituent amino acids included in the proteinisoform A and the protein isoform D. For example, the differentialabsorbance can arise from a difference in the specific amino acids(e.g., amino acids with charged side chains and/or chemical changes tothe charged side chains such as deamidation, glycation, or the like)included in the separated protein isoform A compared to the separatedprotein isoform B.

As shown in FIG. 4B, the fluorescence signal 461 includes peaks 462 witheach peak corresponding to each separated protein isoform having thecorresponding pI value. As an example, a relatively larger peak 462B offluorescence is associated with a protein isoform B with a pI ofapproximately 8.75 at 463B compared to a relatively smaller peak 462D offluorescence that is associated with the protein isoform D with a pI ofapproximately 8.9 at 463D. The differential fluorescence between thepeaks 462B and 462D can indicate a difference in the amount of freethiol groups included in the protein isoform B and the protein isoformD. As described previously, control markers or pI markers can be used,for example to calibrate the method used for separation of proteinisoforms, by generating peaks at known pI values as true positives. Forexample, the peak of absorbance at pI values 7.2 in the absorbancesignal 451 and the peak of fluorescence at pI values 7.2 in fluorescencesignal 461 can correspond to a control marker or a pI marker.

FIG. 5 is a chart 550 showing an overlay of the absorbance signal 451 ofFIG. 4A and the fluorescence signal 461 shown in FIG. 4B. Theexperimental examination of the Herceptin® Biosimilar sample describedwith reference to FIGS. 4A and 4B, also included examination ofdetection binders (e.g., fluorescent dyes) that were unbound orunconjugated with any protein isoforms, also referred to as “dye blank”.In addition to detection and measurement of fluorescence and absorbancesignals associated with protein isoforms, fluorescence and absorbancesignals were also acquired from the dye blank or the detection binders(e.g., fluorescent dyes) that were unbound or unconjugated with anyprotein isoforms to be used as reference signals. FIG. 5 also includesexample reference signals 471 and 481. The reference signal 471corresponds to fluorescence signal acquired from detection bindersunbound to any protein isoforms. The reference signal 481 corresponds toor an absorbance signal acquired from detection binders unbound to anyprotein isoforms. In some implementations, the examination of dye blankto acquire reference signals can be performed independently from theseparation and examination of protein isoforms in a sample (e.g., in aseparate run or session from the run or session involving separation andmeasurement of fluorescence and/or absorbance signals associated withprotein isoforms). In some implementations, the examination of dye blankto acquire reference signals can be performed along with the separationand examination of protein isoforms in a sample, for example byallocating a separate track or capillary for the dye blank such that thedye blank is not exposed to protein isoforms.

As shown, the reference signal 471 lacks discernible peaks indicatingthat the peaks observed in the fluorescence signal 461 are trulyassociated with detection binders that are bound to protein isoforms andare not spurious signal artifacts. The reference signal 481 also isshown to lack specific peaks in intensity showing that the peaksobserved in the absorbance signal 451 are truly absorbance associatedwith protein isoforms and not spurious absorbance artifacts. The plot offluorescence signal 461 and absorbance signal 451 illustrate arelationship between peaks observed in each signal with the peaksobserved in the other signal, the peaks being associated with a set ofplurality of proteins and/or protein isoforms of the plurality ofproteins and/or protein isoforms, as analyzed using a system asdescribed herein, according to an embodiment. Two example pairs of peaksand their correspondence are indicated by arrows.

Free thiols can be used as a critical quality attribute of proteins(e.g., therapeutic proteins) to grade a quality associated with theprotein. A critical quality attributes (CQA) of a clinical product canbe defined as “A physical, chemical, biological, or microbiologicalproperty or characteristic that should be within an appropriate limit,range, or distribution to ensure the desired product quality” Said inanother way products (e.g., therapeutics, monoclonal antibodies (mAbs),etc.) can have identified CQAs (e.g., as a part of implementation ofquality by design (QbD) for development and production ofbiopharmaceuticals) that can include attributes that have an impact onthe clinical efficacy and/or safety of the clinical product. An amountof free thiols in a product can be considered a CQA for productdevelopment. Free thiols can be a leading cause of aggregation, whichcan lead to immunogenicity, thus impacting efficacy. In some instances,free thiols can form during harvesting and/or purification process of atherapeutic protein. In some instances, for example, free thiols can begenerated during the production of bispecific antibodies and/or AntibodyDrug Conjugates (ADCs). Therefore amount of free thiols as determined bythe method described herein using systems described herein can be usedas a CQA for therapeutic proteins.

In some instances, the methods described herein can also be used tocalculate ADC labeling efficiency measurements. In some instances,methods and systems described hereon can be used to quantify bi- and/ormulti-specific antibody reassembly, for example, in conditions involvingsulfhydryl group manipulation. Bi-specific antibodies are generated asindividual half-antibodies, purified, then subsequently exposed to amild reductant to reduce the hinge region to facilitate assembly of thecomplete (or whole) bi-specific antibody. The methods described hereinallow quantitation of free-thiol pre- and post-assembly of bi- andmulti-specific antibodies.

For example, the fluorescent detection binders used to bind with thiolscan be a first set of fluorescent detection binders. Additionally, asecond set of fluorescent detection binders can be used with afluorescence emission in a band of wavelengths different from the bandof wavelengths of light emitted by the first set of fluorescencedetection binders. The second set of fluorescent detection binders canbe configured to bind to a drug included in an ADC such that upon beingexcited by a light (e.g., a light of a single wavelength in the UV range(e.g., 280 nm)) (i) the emission from the first set of fluorescencedetection binders can be used to quantify an amount of free thiolsassociated with the ADC included in each separated protein isoform of aset of separated protein isoforms, and (ii) the emission from the secondset of fluorescence detection binders can be used to measure anantibody-to-drug ratio associated with the ADC included in eachseparated protein isoform of a set of separated protein isoforms. Insome implementations, the systems and/or methods described herein can beused to perform secondary measurements or assessments in addition to thequantification of free and/or total thiol quantification. For example,as the fluorescent detection binders can be configured to have no effector have a controlled effect on the charge heterogeneity of proteinisoforms included in a sample, such that the effect can be accountedfor, the methods used for quantification of thiol content (free and/ortotal thiol groups) also referred to as fluorescent thiolderivatization, may not affect the charge of the un-derivatized isoform.Thus, additional “shift assays” could be developed to further analyzethe protein isoforms. The derivatization of mono-charged ormulti-charged fluorescent dyes could be used isolate a CQA from the restof sample peaks for baseline integration and quantification. Theinclusion of additional charged entities to the labeling reagent couldinduce a significant shift that isolates the peaks of interest (labeledwith dye) away from unlabeled overlapping protein isoform peaks.

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, the term “a member” is intended to mean a singlemember or a combination of members, “a material” is intended to mean oneor more materials, or a combination thereof.

As used herein, the term “sample” refers to a composition that containsan analyte or analytes to be analyzed or detected or used otherwise. Asample can be heterogeneous, containing a variety of components (e.g.,different proteins, different types of protein isoforms, differentcomplexes or conjugates of components like antibody drug conjugates orother components conjugated with proteins etc.) or homogenous,containing one component. In some instances, a sample can be naturallyoccurring, a biological material, and/or a man-made material.Furthermore, a sample can be in a native or denatured form. In someinstances, a sample can be a single cell (or contents of a single cell)or multiple cells (or contents of multiple cells), a blood sample, aplasma sample, a tissue sample, a skin sample, a urine sample, a watersample, etc. In some instances, a sample can be from a living organism,such as a eukaryote, prokaryote, mammal, and/or human.

Devices and/or systems disclosed herein can include any suitableelectronic devices. For example, in some embodiments, instruments caninclude an integral compute device and/or a peripheral compute devicesuch as a personal computer (PC), a personal digital assistant (PDA), asmart phone, a laptop, a tablet PC, a server device, a workstation,and/or the like. The compute device can include at least a memory, aprocessor. In some embodiments, the compute device can an output device,which can be any suitable display that can provide at least a portion ofa user interface for a software application (e.g., a mobile application,a PC application, an internet web browser, etc.) installed on theelectronic device. In such embodiments, the display can be, for example,a cathode ray tube (CRT) monitor, a liquid crystal display (LCD)monitor, a light emitting diode (LED) monitor, and/or the like. In otherembodiments, the output device can be an audio device, a haptic device,and/or any other suitable output device. In some embodiments, thecompute device can include a network interface, which can be, forexample, a network interface card and/or the like that can include atleast an Ethernet port and/or a wireless radio (e.g., a WiFi® radio, aBluetooth® radio, etc.). The memory can be, for example, a random accessmemory (RAM), a memory buffer, a hard drive, a read-only memory (ROM),an erasable programmable read-only memory (EPROM), and/or the like. Theprocessor can be any suitable processing device configured to run orexecute a set of instructions or code. For example, the processor can bea general purpose processor, a central processing unit (CPU), anaccelerated processing unit (APU), and Application Specific IntegratedCircuit (ASIC), and/or the like. The processor can be configured to runor execute a set of instructions or code stored in the memory associatedwith using, for example, a PC application, a mobile application, aninternet web browser, a cellular and/or wireless communication (via anetwork), and/or the like, as described in further detail herein. Thememory can be, for example, a random access memory (RAM), a memorybuffer, a hard drive, a read-only memory (ROM), an erasable programmableread-only memory (EPROM), cloud storage and/or the like. In someembodiments, the memory can be configured to store, for example, one ormore modules that can include instructions that can cause a processor toperform one or more processes, functions, and/or the like.

While various embodiments have been described herein, it should beunderstood that they have been presented by way of example only, and notlimitation. For example, although embodiments described herein involveproteins and/or protein isoforms, it should be understood that methodsdescribed herein are also applicable to other thiol-containingmaterials. Furthermore, although various embodiments have been describedas having particular features and/or combinations of components, otherembodiments are possible having a combination of any features and/orcomponents.

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Where methods and steps described above indicate certainevents occurring in certain order, the ordering of certain steps may bemodified. In some instances, certain steps may be followed while certainother steps may be omitted. For example, although some embodiments aredescribed carrying out fluorescence emission measurements and/orabsorbance measurements on a first portion of a sample containing adetection binder and carrying out fluorescence emission measurementsand/or absorbance measurements on a second portion of the sample thatdoes not contain detection binders, it should be understood that inother embodiments, the sample may not be divided into two portions, adetection binder can be added to the sample, and fluorescence emissionmeasurements and/or absorbance measurements may be carried out only onthe sample containing the detection binder. As another example, in someinstances fluorescence emission measurements can be carried whileomitting UV absorbance measurements or vice versa. Additionally, certainof the events may be performed repeatedly, concurrently in a parallelprocess when possible, as well as performed sequentially as describedabove. For example, detecting fluorescence in a sample well is describedbefore detecting UV absorbance is described. It should be understood,however, that detecting fluorescence and UV absorbance can occur in anyorder or simultaneously. Furthermore, certain embodiments may omit oneor more described events.

Where methods are described, it should be understood that such methodscan be computer-implemented methods. Similarly stated, a non-transitoryprocessor readable medium can store code representing instructionsconfigured to cause a processor to cause the described method to occuror be carried out. For example, an instrument, such as Maurice™ producedand sold by ProteinSimple®, a Bio-Techne® brand, can include a processorand a memory and can cause one or more method steps described herein tooccur. Thus, some embodiments described herein relate to a computerstorage product with a non-transitory computer-readable medium (also canbe referred to as a non-transitory processor-readable medium) havinginstructions or computer code thereon for performing variouscomputer-implemented operations. The computer-readable medium (orprocessor-readable medium) is non-transitory in the sense that it doesnot include transitory propagating signals per se (e.g., a propagatingelectromagnetic wave carrying information on a transmission medium suchas space or a cable). The media and computer code (also can be referredto as code) may be those designed and constructed for the specificpurpose or purposes.

Although various embodiments have been described as having particularfeatures and/or combinations of components, other embodiments arepossible having any combination or sub-combination of any featuresand/or components from any of the embodiments described herein.

1. A method, comprising: generating, from a sample containing aplurality of protein isoforms, a first sample portion and a secondsample portion, at least some protein isoforms from the plurality ofprotein isoforms having one or more free thiol groups; applying, to thefirst sample portion, a fluorescent detection binder configured to bindto free thiol groups to produce conjugated thiols, without reducing theplurality of protein isoforms included in the first sample portion;separating the plurality of protein isoforms in the first sample portionto generate a first set of separated protein isoforms; separating theplurality of protein isoforms in the second sample portion to generate asecond set of separated protein isoforms; detecting a first fluorescencesignal associated with the conjugated thiols and an absorbance signalassociated with the first set of separated protein isoforms;identifying, based on the absorbance signal, at least one of a quantityor an identity of each separated protein isoform from the first set ofseparated protein isoforms; detecting a second fluorescence signalassociated with the second set of separated protein isoforms; andmeasuring, based on a difference between the first fluorescence signaland the second fluorescence signal, a quantity of free thiol groupsassociated with each separated protein isoform from the first set ofseparated protein isoforms.
 2. The method of claim 1, wherein: aquantity of each separated protein isoform from the first set ofseparated protein isoforms is identified, the method further comprising:determining, for each separated protein isoform from the first set ofseparated protein isoforms, a number of free thiol groups associatedwith that protein isoform based on a quantity of protein isoforms forthat protein isoform and a quantity of free thiol groups associated withthat protein isoform.
 3. The method of claim 1, wherein the absorbancesignal is a first absorbance signal, the method further comprising:detecting a second absorbance signal associated with the second set ofseparated protein isoforms, the at least one of the quantity or theidentity of each separated protein isoform from the first set ofseparated protein isoforms is identified based on second absorbancesignal.
 4. The method of claim 1, wherein: at least some proteinisoforms from the plurality of protein isoforms have one or moredisulfide groups; the one or more free thiol groups is a first set offree thiol groups; and the conjugated thiols are a first set ofconjugated thiols, the method further comprising: generating, from thesample, a third sample portion and a fourth sample portion; applying, tothe third sample portion and the fourth sample portion, a reducing agentconfigured to reduce disulfide groups to generate a second set of one ormore free thiol groups; applying, to the third sample portion, thefluorescent detection binder such that the fluorescent detection binderbinds to the first set of free thiol groups and the second set of freethiol groups to produce a second set of conjugated thiols; separatingthe plurality of protein isoforms in the third sample portion togenerate a third set of separated protein isoforms; separating theplurality of protein isoforms in the fourth sample portion to generate afourth set of separated protein isoforms; detecting a third fluorescencesignal associated with the second set of conjugated thiols and a secondabsorbance signal associated with the third set of separated proteinisoforms; identifying, based on the second absorbance signal, at leastone of a quantity or an identity of each separated protein isoform fromthe third set of separated protein isoforms; detecting a fourthfluorescence signal associated with the fourth set of separated proteinisoforms; and measuring, based on a difference between the thirdfluorescence signal and the fourth fluorescence signal, a quantity ofthe first set of free thiol groups and a quantity of disulfide groupsassociated with each separated protein isoform from the third set ofseparated protein isoforms.
 5. The method of claim 4, wherein thequantity of the first set of free thiol groups and the quantity ofdisulfide groups is collectively a quantity of total thiol associatedwith the plurality of protein isoforms, the method further comprising:determining, based on a difference between the quantity of total thioland the quantity of the first set of free thiol groups, the quantity ofdisulfide groups.
 6. The method of claim 1, wherein the plurality ofprotein isoforms in the first sample portion and the plurality ofprotein isoforms in second sample portion are separated via isoelectricfocusing.
 7. The method of claim 1, wherein the quantity of free thiolgroups associated with each separated protein isoform from the first setof separated protein isoforms is a relative quantity, the method furtherincluding: applying the fluorescent detection binder to a standardsample that includes a known quantity of free thiol groups; detecting athird fluorescence signal associated with the known quantity of freethiol groups; and measuring, based on a ratio between the firstfluorescence signal and the third fluorescence signal, an absolutequantity of free thiol groups associated with each separated proteinisoform from the first set of separated protein isoforms.
 8. The methodof claim 1, wherein the plurality of protein isoforms includesantibodies.
 9. The method of claim 1, wherein the first fluorescencesignal and the absorbance signal are obtained simultaneously.
 10. Themethod of claim 1, wherein the plurality of protein isoforms includes aset of drug-antibody conjugates, the method further comprising:quantifying a drug-antibody ratio associated with each drug-antibodyconjugate from the set of drug antibody conjugates.
 11. A system,comprising: a memory; and a processor operatively coupled to the memory,the processor configured to: receive a fluorescence signal associatedwith a first set of separated protein isoforms, the first set ofseparated protein isoforms being obtained from a first portion of asample containing a plurality of protein isoforms, at least some proteinisoforms from the plurality of protein isoforms having one or more freethiol groups, the fluorescence signal being associated with afluorescent detection binder applied to the first sample portion that isconfigured to bind to free thiol groups, receive a first absorbancesignal associated with the first set of separated protein isoforms,receive a second absorbance signal associated with a second portion ofthe sample, identify, based on the first absorbance signal and thesecond absorbance signal, at least a subset of the protein isoforms fromthe sample, calculate, based on the fluorescence signal, a quantity offree thiol groups associated with each protein isoform from the subsetof the protein isoforms.
 12. The system of claim 11, wherein a quantityof each protein isoform from the subset of the protein isoforms isidentified, and the processor is further configured to: determine aquantity of each protein isoform from the subset of the protein isoformsbased on at least one of the first absorbance signal or the secondabsorbance signal; determine, for each protein isoform from the subsetof the protein isoforms, a number of free thiol groups associated withthat protein isoform based on a quantity of that protein isoform and aquantity of free thiol groups associated with that protein isoform. 13.The system of claim 11, wherein the first portion of the sample and thesecond portion of the sample are each separated via isoelectric focusingto obtain the first set of separated protein isoforms and the second setof separated protein isoforms, respectively.
 14. The system of claim 11,wherein the fluorescence signal associated with the first set ofseparated protein isoforms is a first fluorescence signal, and thequantity of free thiol groups associated with each of the subset of theprotein isoforms is a relative quantity of free thiol groups, theprocessor further configured to: receive a second fluorescence signalassociated with a standard sample including a known quantity of freethiol groups; and calculate, based on a ratio between the firstfluorescence signal and the second fluorescence signal, an absolutequantity of free thiol groups associated with each protein isoform fromthe subset of the protein isoforms.
 15. The system of claim 11, whereinthe first portion of the sample and the second portion of the sample areeach separated via isoelectric focusing to obtain the first set ofseparated protein isoforms and the second set of separated proteinisoforms, respectively, the processor further configured to: detect afirst peak associated with the first absorbance signal and a second peakassociated with the second absorbance signal; correlate the first peakto the second peak to generate a matched peak associated with anidentified protein isoform from a set of identified protein isoforms.16. The system of claim 11, wherein the fluorescence signal is a firstfluorescence signal, the processor further configured to: receive asecond fluorescence signal associated with the second portion of thesample, the quantity of free thiol groups associated with each proteinisoform from the subset of the protein isoforms calculated based on adifference between the first fluorescence signal and the secondfluorescence signal.
 17. The system of claim 11 wherein the plurality ofthe protein isoforms includes drug-antibody conjugates, the processorfurther configured to: quantify a drug-antibody ratio associated witheach drug-antibody conjugate.
 18. The system of claim 11 wherein atleast a subset of the protein isoforms includes a plurality ofdrug-antibody conjugates, the processor further configured to: quantifya drug-antibody ratio associated with each drug-antibody conjugate fromthe plurality of drug-antibody conjugates; and calculate a labellingefficiency associated with each drug-antibody conjugate from theplurality of drug-antibody conjugates.
 19. A method, comprising:applying, to a sample containing a plurality of protein isoforms, afluorescent detection binder configured to bind to free thiols withoutreducing protein isoforms; separating the sample to generate a set ofseparated protein isoforms; obtaining a fluorescence signal and anabsorbance signal associated with the set of separated protein isoforms;identifying, based on the absorbance signal, at least one of a quantityor an identity of each separated protein isoform from the set ofseparated protein isoforms; and measuring, based on the fluorescencesignal, a quantity of free thiols associated with each separated proteinisoform from the set of separated protein isoforms.
 20. The method ofclaim 19, wherein a quantity of each separated protein isoform from theset of separated protein isoforms is identified, the method furthercomprising: determining, for each separated protein isoform from the setof separated protein isoforms, a number of free thiol groups associatedwith that protein isoform based on a quantity of protein isoforms forthat protein isoform and a quantity of free thiol groups associated withthat protein isoform.
 21. The method of claim 19 wherein the quantity offree thiol groups associated with each separated protein isoform fromthe set of separated protein isoforms is a relative quantity, and thefluorescence signal is a first fluorescence signal, the method furtherincluding: obtaining a second fluorescence signal associated with astandard sample including a known quantity of free thiol groups; andmeasuring, based on a ratio between the first fluorescence signal andthe second fluorescence signal, an absolute quantity of free thiolgroups associated with each separated protein isoform from the set ofseparated protein isoforms.